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RNAi-mediated suppression of endogenous storage proteins leads to a change in localization of overexpressed cholera toxin B-subunit and the allergen protein RAG2 in rice seeds

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Abstract

Key message

RNAi-mediated suppression of the endogenous storage proteins in MucoRice-CTB-RNAi seeds affects not only the levels of overexpressed CTB and RAG2 allergen, but also the localization of CTB and RAG2.

Abstract

A purification-free rice-based oral cholera vaccine (MucoRice-CTB) was previously developed by our laboratories using a cholera toxin B-subunit (CTB) overexpression system. Recently, an advanced version of MucoRice-CTB was developed (MucoRice-CTB-RNAi) through the use of RNAi to suppress the production of the endogenous storage proteins 13-kDa prolamin and glutelin, so as to increase CTB expression. The level of the α-amylase/trypsin inhibitor-like protein RAG2 (a major rice allergen) was reduced in MucoRice-CTB-RNAi seeds in comparison with wild-type (WT) rice. To investigate whether RNAi-mediated suppression of storage proteins affects the localization of overexpressed CTB and major rice allergens, we generated an RNAi line without CTB (MucoRice-RNAi) and investigated gene expression, and protein production and localization of two storage proteins, CTB, and five major allergens in MucoRice-CTB, MucoRice-CTB-RNAi, MucoRice-RNAi, and WT rice. In all lines, glyoxalase I was detected in the cytoplasm, and 52- and 63-kDa globulin-like proteins were found in the aleurone particles. In WT, RAG2 and 19-kDa globulin were localized mainly in protein bodies II (PB-II) of the endosperm cells. Knockdown of glutelin A led to a partial destruction of PB-II and was accompanied by RAG2 relocation to the plasma membrane/cell wall and cytoplasm. In MucoRice-CTB, CTB was localized in the cytoplasm and PB-II. In MucoRice-CTB-RNAi, CTB was produced at a level six times that in MucoRice-CTB and was localized, similar to RAG2, in the plasma membrane/cell wall and cytoplasm. Our findings indicate that the relocation of CTB in MucoRice-CTB-RNAi may contribute to down-regulation of RAG2.

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Abbreviations

BiP:

Binding protein

CTB:

Cholera toxin B-subunit

DAF:

Days after flowering

IF:

Immunofluorescence

immuno-EM:

Immuno-electron microscopy

RNAi:

RNA interference

PB-I:

Protein body I

PB-II:

Protein body II

PBS:

Phosphate-buffered saline

SDS-PAGE:

SDS-polyacrylamide gel electrophoresis

T-DNA:

Transfer DNA

WB:

Western blotting

WT:

Wild type

Ab:

Antibody

mAb:

Monoclonal antibody

BSA:

Bovine serum albumin

NCS:

Newborn calf serum

PSV:

Protein storage vacuole

ER:

Endoplasmic reticulum

RER:

Rough endoplasmic reticulum

IL-10:

Interleukin-10

PEG:

Polyethylene glycol

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Acknowledgments

We are grateful to Drs. Nana Kawasaki, Kunisuke Tanaka, Koji Kashima, Michiyo Abe, Yoshiko Fukuyama, Yuhi Saito, and Takanari Shigemitsu for useful discussions and technical support. This work was supported by grants from the Programs of Special Coordination Funds for Promoting Science and Technology and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Y.Y., H.K.); the Ministry of Health, Labor and Welfare of Japan (Y.Y., H.K.); the New Energy and Industrial Technology Development Organization (NEDO) (H.K.); the Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-step) and the Research and Development Program for New Bio-industry Initiatives of the Bio-oriented Technology Research Advancement Institution (Y.Y.).

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Authors and Affiliations

  1. Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan

    Shiho Kurokawa, Mio Mejima, Yuko Takahashi, Natsumi Takeyama, Hiroshi Kiyono & Yoshikazu Yuki

  2. Laboratory of Genetic Engineering, Graduate School of Life and Environmental Science, Kyoto Prefectural University, 1-5, Nakaragi-cho, Shimogamo, Sakyo-ku, Kyoto, 606-8522, Japan

    Shiho Kurokawa, Shigeru Satoh & Takehiro Masumura

  3. Crop Development Division, NARO Agricultural Research Center, 1-2-1, Inada, Joetsu-shi, Niigata, 943-0193, Japan

    Masaharu Kuroda

  4. National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo, 158-8501, Japan

    Rika Nakamura & Reiko Teshima

  5. Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan

    Hiroshi Sagara

  6. Biotechnology Research Department, Kyoto Prefectural Agriculture, Forestry and Fisheries Technology Research Center, 74, Kitainayazuma, Ohji, Seika-cho, Soraku-gun, Kyoto, 619-0244, Japan

    Shigeru Satoh & Takehiro Masumura

  7. International Research and Development Center for Mucosal Vaccine, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan

    Hiroshi Kiyono & Yoshikazu Yuki

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  1. Shiho Kurokawa
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  2. Masaharu Kuroda
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  4. Rika Nakamura
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  5. Yuko Takahashi
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  6. Hiroshi Sagara
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  7. Natsumi Takeyama
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  8. Shigeru Satoh
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  9. Hiroshi Kiyono
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  10. Reiko Teshima
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  11. Takehiro Masumura
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  12. Yoshikazu Yuki
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Correspondence to Yoshikazu Yuki.

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Communicated by F. Sato.

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Kurokawa, S., Kuroda, M., Mejima, M. et al. RNAi-mediated suppression of endogenous storage proteins leads to a change in localization of overexpressed cholera toxin B-subunit and the allergen protein RAG2 in rice seeds. Plant Cell Rep 33, 75–87 (2014). https://doi.org/10.1007/s00299-013-1513-3

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  • DOI: https://doi.org/10.1007/s00299-013-1513-3

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